Gram positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
Various gram positive microorganisms are known to secrete extracellular and/or intracellular proteases at some stage in their life cycles. Some of these proteases are produced in large quantities for industrial purposes. However, a negative aspect of the presence of proteases in gram positive organisms is their contribution to the overall degradation of secreted heterologous or foreign proteins.
The classification of proteases found in microorganisms is based on their catalytic mechanism which results in four groups: serine proteases, metalloproteases, cysteine proteases, and aspartic proteases. These categories can be distinguished by their sensitivity to various inhibitors. For example, serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); metalloproteases by chelating agents; cysteine proteases by iodoacetamide and heavy metals and aspartic proteases by pepstatin. Further, in general, serine proteases have alkaline pH optima, metalloproteases are optimally active around neutrality, and cysteine and aspartic proteases have acidic pH optima (Biotechnology Handbooks, Bacillus. Vol. 2, edited by Harwood, 1989, Plenum Press, New York).
Metallo-proteases form the most diverse of the catalytic types of proteases. Family M20b is also known as the glutamate carboxypeptidase family and includes bacterial enzymes such as the glutamate carboxypeptidase from Pseudomonas spp., succinyldiaminopimelate desuccinylase (DAPE) from E. coli and acetylornithine deacetylase (ARGE) also from E. coli (Methods in Enzymology, vol. 248, Academic Press, Inc. 1994). Preferred metal ions for the enzymes of the M20b family include zinc and/or cobalt.